Analysis of fats 5md oils by SFE and SFC

upercritical fluids (SCF) have attracted much attention over the past 20 years with regard to their potential application in chemical engineering, industrial processing and environmental remediation. These fluids, patiicularly supercritical carbon dioxide (SC-CO,), permit extraction and processing operations to be conducted at relatively low temperatures. using nontoxic and inert gases. The resultant products (both extract and substrate) from these SCF-based processes are solvent-free and minimally altered or degraded during the extraction process. Such factors have served as the basis for the special applications of these fluids in the food industry (1). Since the early 198Os, there has been a renewed interest in the analytical applications of SCF (2). This has largely been due to the development of suitable analytical equipment for

conducting supercritical fluid chromatography (SFC) and supercritical fluid extraction (SFE) on a routine basis (3). Analytical SFE and SFC continue to be developed to meet the widespread demands of many analysts in the food, environmental and energy-related industries (4). These developments are accelerated by new government regulations regarding the

2(X)

3(W)

J(K)

5lXI
Irc.\\LIrc I;11111 1

h(K)

7(K)

XW

Figure

1. Solubiliiy

01 soybean

oil triglycerides

in SC-CO,

as a function

of pressure

generation, use and disposal of hazardous solvents in the laboratory environment (5). Regardless of the scale of the SFE or SFC operation, certain fundamental principles apply. An SCF can be viewed as a unique state of matter, intermediate between a liquid and a gas, whose physical properties are determined by the external pressure and temperature that are applied to the fluid. If the fluid is held at a temperature and pressure above its critical point (T, and PC, respectively), then it is said to be in the SCF state, and its density under such conditions can be varied substantially by increasing the applied pressure on the system. Suffice it to say that at high densities such fluids take on the solvent-like properties of many organic solvents and have the capability to dissolve a variety of substances, just as normal liquids do. Why then should SCFs be of particular interest to the fats and oils analyst? The answer lies partly in the extraordinarily high solubility exhibited by lipid materials in SCF, particularly CO*, which readily solubilizes nonpolar solutes (6). Such a trend is illustrated by Figures I and 2, where the solubility of soybean oil triglycerides as a function of CO2 pressure (7) is plotted. For the illustrated isotherms, it is possible to obtain lipid solubilities ranging from a few weight percent to over 25 wt%. depending on the pressure and temperature condtions chosen. Such solubilities arc more then sufficient for chromatography under SCF conditions. the cnh;luslivc dclipidalion of fat- and oilcont;Iining samples hy SFE. and lhc
INFORM. Vol. 4. no 9 (September 1993)

1090

INSTRUMENTATION

ocoplirrols. I( ii,tr solubility

Other lipids. such as fatty acids, sterols. etc.. exhibit simitrends as those depicted iln Figures 1 and 2 (8.9). Unfortunately tlhe. high overall solubility of many Iipid compounds in SCF compromises IIhe molecular specificity of SFE in the absence of an auxiliary techntque, Such as chromatography (IO). Howeve:r. the use of lower pressures and/or t emperatures permits SCF to be applied to many analytical upplica3 t ions that do not require such high finite lipid solubilitics, such as capilIat-y SFC. residue analysis and on-line :jFE. We shall now examine some of t hese applications to illustrate the usefulness of analytical SFE and SFC in applied lipid analysis. tXl-line SFE tiechanistically, analytical SFE is 1 itpplied in either an off- or on-line Inode. Off-line extraction usually i mplies that the sample of interest is :xtracted in a discrete operation in c !,vhich the extract is first isolated and 1hen independently analyzed by any ()ne of a variety of techniques. Within Ilimits. the extraction and temperature and pressure can be varied to control I:he composition of the lipid extract; Inowever. it is common when extractIing lipid matter from different sample Imatrices to do an exhaustive extracItion. However, even when perfotming ,extractions at high pressures and temperatures (700 bar, 8OC), an excellent separation can be achieved between phospholipids and nonpolar lipids (I 1). The former can easily be solubilized in SCF by the additj.on of a cosolvent, such as ethanol or methanol to the SC-CO2. An example of a selective extraction of interest to the lipid analyst is the isolation of cholesterol from oil or fat matrix, such as cod liver oil matrix (12). Adjustment of the CO2 density to 0.40 g/mL (120 atm, 60C) allows the cholesterol to be isolated from the oil as shown in Figure 3. A higher CO2 density, 0.93 g/mL (350 atm, 40C). permits extraction of the triglycerides, as indicated by the upper

IO

11

12

I.;

Pressure x 100 (bar)

Figure 2. Solubility of soybean oil triglycerides in SC-CO2 at high pressure

INFORM, Vol. 4, no. 9 (September

1993)

1092

INSTRUMENTATION
,cck :II~ alternative
:ancicnt) zxtrsction IO

to the classic
Soshlct

and oltcn-varlcd

3 2

!
2 4 Time (min)

Triglycerides/fats

Figure

3. HPLC of SFE fractions

of cod liver oil

technique. which utilizes Drganic and sometimes flammable and :arcinogenic solvents. There remains little doubt that analytical SFE can yield equivalent results to extractions nn the same sample using nonpolar organic solvents. This recently has been demonstrated by researchers at NCAUR for the quantitative extraction of oil from three different oilseed 1ypes( 14). Perhaps of more interest are the recent results for the extraction of fat from different food matrices by Hopper (as cited in 15) as noted in Table I. In this case, the SFE results were determined by a simultaneous multisample SFE. using a instrument designed for large samples that is a prototype of an earlier unit developed by researchers at NCAUR (13). Note that the SFE results from this off-line technique using high pressure CO? are comparable to two methods using liquid solvents. Method 960.39 is a Soxhlet extraction using petroleum ether as specified by the AOAC International (16), while the results in the column labeled PAM 1 are a sequential solvent extraction using ethyl ether, as designated in the Pesticide Analytical Manual of the FDA (17). Despite these encouraging results, collaborative studies need to be undenaken to verify the reproductivity of the SFE method, particularly in lieu of its importance in nutrient analysis ( 18). On-line SFE In contrast to off-line SFE, on-line SFE involves conducting an extraction, followed by transfer of the extract to the analysis instiument, all in a sequential fashion. The analysis instrument of choice is frequently a gas or liquid chromatograph, followed by mass or infrared spectrometry for identification of the separated compounds. The technique has certain advantages and disadvantages, which are worth enumerating. On-line SFE frequently requires the use of switching and sampling valves to transport the extract from the extraction stage to the analysis step. The extract is deposited and concentrated during a defined period of

(conrimrcdfrompage

1090)

ultraviolet detector trace (210 nm) from high-performance liquid chromatographic analysis of the two estracts (Fig. 3). This simple fractionation was accomplished on a HewlettPackard Model 7680A SCF extractor, using a CO2 flow rate of 4 mL/min for 10 min. after an initial static hold of 1 min. The cod liver oil sample (500 FL) was initially mixed with a diatomaceous earth sorbent. Off-line SFE preparation and analysis of multiple samples is currently available, due in part to the initial pioneering efforts of researchers at NCAUR (13). Rapid SFE (15 min) of lipid phases in pesINFORM, Vol. 4, no. 9 (September 1993)

ticide-containing meat products could be affected simultaneously in six samples by using the apparatus shown in Figure 4. Quantitative lipid and pesticide extractions were obtained at 340-680 atm at 60C using 5-10 L/min CO2 flow rates (ambient conditions). Today, commercial instrumentation exists that will permit the analysis of up to 8, 24 or 44 samples, either simultaneously or in a serial mode of extraction. One particular application of offline SFE deserves special mention: the determination of fats and oils levels in raw materials and/or processed food products. This application is becoming critically important as analysts

1093

Figure 4. multi-sari extractor

AUR SCF

extraction, either on some form of a retention gap or at the head of the chromatographic column. This prevents contamination of and loss of the extract prior to the analysis step. However, the analyst employing on-line SFE also loses the freedom IO choose the analytical method once the extraction module ih fixed in the system. Considerable replumbing may be necessary IO mate the SFE step with an alternative analytical technique. The sample sizes that can be analyzed

by on-line SFE are often small (mg) in the case of lipid-containing substrates, since the high solubility of lipid compounds in the SCF tends to lead to column overloading in the case of microbore and capillary columns. The careless handling of extraction cells can also lead to analysis artifacts, such as lipids from fingerprints, which show up on the resultant chromatograms. However. the ability of on-line SPE to extract small samples for subsequent analysis is also an attractive

Table 1 Percent

fat extracted

(%RSD,

n = 6)

Pork snusafe

Pc;inul hurter
Cheddsr cheese

Corn chip

30.s5 so.32 33.8s il.32

(4.7.5) (0.39) (3.13) (0.62)

29.83 49.29 33.94 3 I .x0

( I .55) (0.60) t.: 16) (0 76)

29.83 (I .32) 492 I (0.44) 33.X11.14) 3 I.5 I (0.46)

1094

INSTRUMENTATION
2.

$*I\ ~lil-olll~iltrgra1,11.

Gcnllc

c\Ir.icIIon

1 pressure 1

CC-MS

Figure

5. Schematic

of SFElGClhlS

system

for volatile

analysis

feature of the technique. King (19) has shown that the lipid content of single seeds and insects can be characterized by coupling on-line SFE with SFC. Volatiles and semi-volatile components from the degradation of fats and oils also can be identified and quantitated by cdupling on-line SFE with SFC or gas chromatography. Recent studies by Snyder (20) have shown

that low temperature/pressure extraction of volaliles/semi-volatiles from as little as IO J.ILof oxidized oil offers an improved method of characterizing the oil decomposition products. The experimental apparatus is very simple. as depicted in Figure 5, consisting of a high pressure syringe pump in-line with a thermostatted macro-extraction cell connected to the injection part of

at I02 ;itm ;III~I 50C limit5 rhc cxlr;ic11011 high nioIccuI;ir wci$il compo01 ncnts and minimizes the dccornposltion of hydraperoxides to lower molecular weight artifacts. thcrebl providing ;I more accurate analysis of volatile components in the osidizcd oil. Recently on-line SFE has been combined with :I precolumn chemical reaction followed by gas and/or SFC for the analysis of reaction products. Such ;I technique allows the study 01 reactions in SCF media. bur pcrhapb more importantly. the anslyssl~ot analytically-useful derivatives. Berg and co-workers (3 I) have synthesized both methyl and butyl esters from interesterification of triglycerides In cdiblc fats using an immobilized lipase in the extraction cell at 1.50 atm and 50C. Similar analytically-useful results have been obtained by King and co-

INFORM,Vol. 4. no. 9 (September 1993)

1095

workers (22), using an aluminum oxide sorbent in the extraction cell for the production of methyl esters. Partial conversion to methyl esters was obtained at 200 atm and 40C on single plant seeds. thereby allowing seed viability to be maintained after on-line analysis of the methyl esters by gas chromatography. SFC SFC offers the lipid analyst some very interesting options that are not easily achieved by using other types of chromatography. Unfortunately SFC is often perceived as a technique that is applicable to only a few niche applications that cannot be solved by gas chromatography (GC) or high-performance liquid chromatography (HPLC). This is not true when one examines the versatility of SFC in applied lipid analysis, as well as advantages of the technique itself. The uniqueness of SFC-based separations derives in part from the ability of the analyst to vary the mobile phase solvating power as a function of pressure. Hence, many separations in SFC are affected in a similar manner to gradient elution techniques in HPLC, where retention and separation are altered by varying the composition of the mobile phase. SFC utilizes both capillary and packed columns for lipid analysis (23). the latter option being capable of producing very high column efficiencies (24) by using ultrasmall diameter columns. Flamc-ionization detection has been the most successful method to date in the analysis of lipids by SFC; howcvcr. rcccnt advances in coupling the evaporative light-scattering dctcctor with SFC have been successful (7-S). Both dctcctars also offer ;I universal mode of detection that is not readily available with HPLC. Several advantages attend the use of SFC that arc missing in GC and HPLC. The use of mobile phase pressure/density-programming techniques can eliminate the need for sample preparation prior to analysis (26). since unwanted or interfering component> can be injected along with the t;lrfct analyses and simply eluted out of ~olunin by increasing the density 01 II~C niohii~ phase. The rclarivcl,

benign conditions employed in SFC make it a technique that is compatible for the chromatography of nonvolatile, but thermally labile compounds, or moieties prone to oxidation. The ability of SFC to analyze lipid-type compounds approaching 1000 Daltons in molecular weight also eliminates the need for derivatization, as required in many GC-based methods. SFC also eliminates or reduces the use of solvents relative to HPLC methods. What are the unique applications of SFC that are of interest to the applied lipid analyst? As noted above, SFC often can be directly applied to an analysis situation without resorting to sample preparation or derivatization.Generic applications include the direct characterization of raw materials or reaction mixtures, the deformulation of commercial products containing a wide range of lipid types and the direct detection of product adulteration or deterioration. Examples of these applications have been provided by King (19). SFC is also unique in its ability to separate oligomeric mixtures of polymers, surfactants and other homologous series of compounds. The high resolving power of capillary SFC for these applications is due in part to the analysts ability to specify complex

pressure, density or temperature programs which facilitate the separation of oligomeric mixtures. Figure 6 demonstrates this type of separation by SFC for an oleic acid-esterified propoxylated glycerol having 5 moles of propylene oxide/mole of glycerol (low caloric fat substitute), which was obtained by using a asymptotic programmed density ramp from 0.12 to 0.6 1 g/mL (27). Other analysts have also employed SFC to great advantage for the characterization of surfactant mixtures (28) and synthetic oligomers (29). Chester (30) recently advocated even higher pressures in SFC to allow the separation of the higher molecular weight species in a oligomeric mixture. Applying this concept along with the choice of the right stationary phase results in an optima1 separation of the higher oligomers in a synthetic mixture of ethoxylated steryl alcohol oligomers (Brij 78). as shown in Figure 7. As in SFE, SFC can provide a general assessment of the lipid components in a natural product matrix, either by coupling SFE with SFC or by simply performing a solvent extraction on the sample matrix, followed by injection into the SFC. An
(continued on page 1097)

2.00

4.00
Tlmc x IO min

6.00

X.00

Figure6. SFCof oleic acid-esterified propoxylatedglycerol

lene oxide/mole of glycerol

(EPG) with 5 moles

of propy-

INFORM,Vol 4. no 9 (September 1993)

1OG7

conrinued from page 1095)

Il. :

Methyl Column. IhOT

1, Biphenyl Column. : ,lIl 200 C

E30

E16,
Cyanopropyl Column.

I20 I 0 I

I80 I I2

240 I

300

360

420

4X0 I

540 I 4x

600 I

660 am J
h0 min

PVXWW I I I 3h 24
Tim

Figure 7. Advantage of high pressure for the SFC analysis Time axis is for the biphenyl chromatogram. 300

of Brij 78.

example of this type of analysis as applied to a study on the absorption of dietary fats is illustrated in Figure 8, where the lipid components in hamster feces have been separated by SFC (31). The separation depicted in Figure 8 was accomplished by superimposing both a temperature and pressure gradient during the SFC run to effect a better separation between the sterol esters and triglycerides. Similar SFC profiles could be obtained from either making a liquid injection of a Soxhlet extract or by inserting a fete pellet into an on-line WE module attached to the SFC. Maturity of an analytical technique can often be assessed by its application to quantitative or collaborative types of analysis. Routine and standard methods based on SFC have emerged recently and offer not only improvements in analytical methodology, but a reduction in solvent disposal or regeneration costs. Recently the status of the official AOCS method for a-monoglycerides has been noted in INFORM (32) and alternative methodology suggested. SFC also can be applied for monoglyceride determination, and recent quantitative studies on commercial emulsifiers indicate that excellent results can be obtained. Table 2 compares the results for total monoglycerides in a commercial emulsifier determined by HPLC using evaporative light-scattering detection. GC of the propionyi ester and SFC with flame-ionization detection on the underivatized sample (33). The agreement between all three methods is excellent. Future horizons The future application and potential of SFE and SFC appears promising, since regulatory concerns involving the use and disposal of hazardous solvents opens up a new vista for the above techniques. The above uses also will be accelerated by the federal Nutritional Labeling and Education Act of 1990, where concern over the lipid content of food will assure new uses for SFE and SFC. On the horizon arc some new applications 01 SCFs. which contarn
INFORM. Vol 4. no 9 (September 1993)

0 Twnc (mln) 0 Pressure calm)75 20 175 30 175 240 own lcnlp. (C) 140 hamster feces (with

Figure 8. SFC separation of the lipid extract from freeze-dried permission of Journal of High Resolution Chromatography).

1098

INSTRUMENTATION
Table 2 Comparison determined TCchllical of monoglyceride results for a commercial by the HPLC-ELSD, GC and SFC method+ Total HPLC-ELSD Lor IX40 emulsifier as Intorm;~~~or~ ScrvIcc.

monoglycerides,d/l00 g
(Xlderivatized SFC/underivatized

Springfield. Virginia. 19x9. Mc[hads 211.13 (C). (1-land (h). 18. Anonymous. INFORM 3.562 ( 1993). t 9. King. J .w.. ./. c/lW~WJfO,~,-. SC;
28.9 ( I YYO).

Mean
%RSD (n) Lot 6022

92.5
I.1 (4)

93.3
1.3 (4)

93.4
3.5 (II )

Mean
%RSD (n)

94.1
I.5 (4)

94.0
I .8 (4)

95.9
3.4 (12)
devi-

uAbbrcvmlwn\. HPLC-ELSD. bxgh-perlormancs tquid chrom~lo~rilphy-cvaporativc light-mnering dcwcmr: CC. gas chronmo~raph~. SFC. wpercnlmt fluid chromnrographyr RSD. relaive standard ~,Wll

elements of both analytical and process technologies. SFE techniques will undoubtedly be coupled with numerous forms of chromatography for further fractionation of the derived extracts and with techniques such as immunoassay (34) for the rapid screening of many samples. The renaissance of SFC indicates the increased use of packed-column methodology, for both capillary and microbore columns, as well as for enhanced selectivity using modest cosolvent addition. References 1. Rizva, S.S.H., J.A. Daniels, A.L. Benado and J.A. Zoliweg Food Tech. 40 (7): 57 (1986). 2. Lee, M.L., and K.E. Markides, Analytical Supercritical Fluid Chromarography and Extraction, Chromatography Conferences Inc., Provo, Utah. 1990. 3. Wenclawiak. B., Analysis with Super-critical Fluids: Extraction and Cht-onlatography, SpringerVerlag, Heidelberg, 1988. 4. Hawthorne, S.B., Anal. Chem. 62 (11):633A (1990). 5. Katauskas, T., and H. Goldner, Res. Del,. 33(4):40 ( I99 1). 6. Stahl, E., K.W. Quirin and D. Gerard, Dense Gases for Extraction and Refining, Springer-Verlag, Heidelberg, 1988, pp. 85-94. 7. Friedrich. J.P., U.S. Patent 4.466. 923 ( 1984).
INFORM, Vol. 4, no. 9 (September 1993)

8. King, J.W., J. Am. Oil Chem. Sot. 601711 (1983). 9. Chrastil, J., J. Phys. Chem. 86:3016 (1982). 10. Favati, F., and J.W. King, Abstracts of the 4th International Symposium on Supercritical Fluid Chromatography and Extraction, Cincinnati, Ohio, May 2&22, 1992, pp. 7 l-72. 11. Friedrich, J.P., G.R. List and A.J. Heakin, J. Am. Oil Chem. Sot. 59:288 (1982). 12. Gere, D.R., L.G. Randall, CR. Knipe, W. Pipkin and L.C. Doherty, Proceedings of the 9th Annual Waste Testing and Qualiry Assurance Symposium, Alexandria, Virginia. July 12-16, 1993. 13. King, J.W.. J.H. Johnson, S.L. Taylor, W.L. Orton and M.L. Hopper, Abstracts of Pittsburgh Conference on Analytical Chemistry and Applied Spectroscopy. Chicago, Illinois, March 4-8, I99 I. Abstract No. 180. 14. Taylor, S.L.. J.W. King and G.R. List, J. Am. Oil Chem. Sot. 70:437 ( 1993). 15. Sawyer, L.D., J. Assoc. Off. Anal. Chem. 76(1):144 (1993). 16. McNeal, J.E. in Oficial Methods of Analysis of the AOAC-15th Ed., Vol. 2. edited by K. Helrich, AOAC International, Arlington, Virginia, 1990, Method 960.39, pp. 93 l-932. 17. Pesticide Analytical Manual (PAM)--FDA, Vol. 1. National

20. Snvdcr. J.M.. INFORAI 4.533 ( 19-93). 21. Berg. B.E.. E.M. Hansen and T. Greibrokk, Abstracts of the 2nd EII~O~CJUS~mposirtn~ on Supe-critical Fluid Chronlaro,~rul)lI! and Extracrion. Huethig-Verlag. Heidelberg. 1993. pp. I%-202. 22. King. J.W.. J.E. France and J.h4. Snyder, Frcsenius J. Anal. CIwn. 334: 474 ( 1992). 23. Markides. K.L., and M.L Lee. SFC Applications, Brigham Young University Press. Provo, Utah, 1988, 1989. 24. Chester, T.L., in Analytical Instrumentation Handbook, edited by G.W. Ewing. Marcel Dekker inc., New York. 1990, pp. 843-88 I. 25. Demirbuker, M.. Analysis of Lipids by Supcrcritical Fluid Chromatography, Ph.D. thesis, Stockholm University. 1992. 26. King, J.W., J. Chromatoqr. Sci. 27:355 (1989). 27. Lu. X.J., M.R. Myers and W.E., Artz, J. Am. Oil Chem. Sot. 70.355 (1993). 28. Silver. A.H., and H.T. Kalinoski. Ibid. 69:599 ( 1992). 29. Geissler, P.R., and A.E. Johnson Jr., Ibid. 67:54 I (1990). 30. Chester, T.L., and D.P. Innis, Abstracts of rite 2nd European Syntpckium on Analytical Supercritical Fluid Chromatography. Huethig-Verlag, Heidelberg, 1993, pp. 16-26. 31. Pinkston, J.D., T.E.Delaney. D.J. Bowling and T.L. Chester, J. High Resolut. Chromtogr. 14:401 (1991). 32. Steiner, J., INFORM 4:706 ( 1993). 33. Liu, J., T. Lee, E. Babik, Jr., M. Guzman-Harty and C. Hastilow, J. Am. Oil Chem. Sot. 70:343 (1993). 34. France, J.E., and J.W. King, J. Assoc. Off. Anal. Chem. 74 (6j.1013 (1991). n

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